RNA silencing refers to a group of widespread gene-regulatory pathways deeply rooted in nearly every facet of human biology, including brain development, stem-cell and germ-line maintenance and cancer progression. At the molecular level, all RNA silencing pathways, such as the microRNA (miRNA) regulatory pathway, are mediated by a specialized family of RNA-binding proteins named Argonaute. Argonaute proteins are uniquely capable of binding small regulatory RNAs and using the encoded sequence information to locate and silence complementary target RNAs. The versatility and power of RNA silencing arises from the fact that Argonaute can be loaded with a small RNA of any sequence and thus can be programmed to target any RNA for silencing. However, despite the importance of Argonaute in human biology and the as-of-yet untapped therapeutic potential, a detailed structural understanding of Argonaute is lacking. Indeed, information relating the structure of Argonaute to its functions is limited, derived either from distant, bacterial forms of the enzyme, which employ DNA guide molecules, or from isolated domains of eukaryotic forms. The contribution of the proposed research will provide high- resolution structures and functional analyses of full-length human Argonaute2 (Ago2). The overarching goal of this proposal is to understand three key interactions of Ago2 on a detailed structural and mechanistic level: (1) guide RNA binding; (2) target RNA recognition; and (3) the binding of the necessary accessory factor TNRC6. In preliminary work, the structure of Ago2 has already been determined and will serve as the launching platform for the studies proposed here. In Aim 1, a combination of structural and biochemical approaches will be used to determine how Ago2 binds guide RNAs and positions them for efficiently identifying target RNAs. This work will provide structural insights for the rational design of improved siRNAs. In Aim 2, a similar approach will be taken to understand the mechanism by which Ago2 recognizes target RNAs. These studies will provide a structural basis for the empirical miRNA targeting rules developed by other labs and open new avenues for manipulating these RNAs for both research and therapeutic purposes. In Aim 3, the structural basis for the association of Ago2 with TNRC6 will be determined. These results will define the general principles that guide the assembly of the massive complexes required for RNA silencing in vivo. Combined, these structural and functional studies will provide comprehensive mechanistic insights into one of the most fundamental-but poorly understood-components of RNA silencing in humans.